Project Summary Head and neck squamous cell cancer (HNSCC) is the sixth most common cancer worldwide and almost 50,000 men and women will develop HNSCC this year in the US alone. Unfortunately, only two-thirds of these HNSCC patients will survive more than five years, a statistic that has not changed in decades despite advances in surgery, radiation therapy, and chemotherapy. Given the well-known co-morbidities and recurrence rates associated with conventional treatments, there is a real need for innovative new approaches to treating HNSCC. Cancer immunotherapy (such as FDA-approved immune checkpoint antibodies) has arisen as an exciting treatment modality for several advanced-stage cancers including HNSCC, with the potential to generate specific and durable anti-tumor responses, although typically only in about 15-20% of patients. Recently, a new class of immunotherapeutics based on synthetic cyclic dinucleotides (CDN) have been found to produce strong anti-tumor responses in preclinical models through the Stimulator of Interferon Genes (STING) pathway. However, to date CDN monotherapy has shown minimal efficacy in preclinical models of HNSCC, requiring multiple-injections and concomitant administration of immune checkpoint antibodies targeting the PD-1/PD-L1 axis for tumor rejection. In response to this challenge, our lab has developed a novel peptide nanofiber hydrogel-based platform for intratumoral CDN delivery called ?STINGel? which has shown impressive and surprising efficacy in a challenging murine model of HNSCC. Our goal is to determine the underlying mechanisms driving STINGel effectiveness in HNSCC. The overall hypothesis of this proposal is that intratumoral injection of STINGel renders immunologically refractory tumors sensitive to immune-mediated killing through multiple mechanisms including: 1) Prolonged release of CDN, 2) STINGel-driven recruitment of critical antigen presenting cells (APCs) to the site of injection, and 3) enhanced Type-1 interferon (IFN) response in both tumor cells and APCs within the tumor microenvironment (TME). We propose to assess the underlying mechanisms for this unique combinatorial immunotherapy in two aims: Aim 1 will examine CDN release kinetics and recruitment of critical APCs into the biomaterial, testing the hypothesis that STINGels can achieve controlled release while also inherently attracting APCs. Aim 2 will assess the ability of STINGel to modify the TME, testing the hypothesis that STINGel recruits increased APCs and induces higher upregulation of the Type 1 IFN pathway in both tumor cells and APCs versus CDN alone, due to enhanced cell uptake of CDN. Overall, these studies are anticipated to enhance our understanding of the mechanisms by which STINGel can reverse resistance to immunomodulatory monotherapy in solid tumors and provide insights into how the adverse tumor microenvironment can be rendered more susceptible to immunotherapy.